The immune system has pivotal influence in the evolution and progression of many tumor types, including colorectal cancer (CRC). In particular, the presence of a strong T cell response in CRC, indicating activation of the adaptive immune system, has been associated with better patient outcomes. As such, recently developed immunotherapeutic approaches often attempt to harness the adaptive immune response. Immune cells are an integral component of the tumor microenvironment, and dynamically interact with neoplastic cells. However, our understanding as to the complexity of the T cell response and the factors that drive this response remains limited. The objective of this proposal is to identify genetic, lifestyle, and tumor factors associated with the T cell response in CRC, and to characterize the survival implications of that response. Specifically, in Aim 1 we will examine the relationship of personal characteristics with T cell response in CRC, including the role of (1a) germline genetic variation within human leukocyte antigen (HLA) and killer-cell immunoglobulin-like receptor (KIR) genes, and (1b) lifestyle factors (e.g., aspirin use, smoking, alcohol consumption). In Aim 2 we will focus on several colorectal tumor characteristics as they relate to T cell response, including (2a) the presence of Fusobacterium nucleatum and bacterial toxin genes in CRC, and (2b) somatic mutations in key signaling pathways (e.g., WNT signaling and RAS/RAF). In Aim 3, we will evaluate the associations of different aspects of T cell response with CRC survival, accounting for known prognostic factors and the relationships identified in Aims 1-2. To achieve these Aims, we propose to assess the density and spatial distribution of specific T cell subsets using multiplexed immunofluorescence (mIF) to quantify expression levels and co-expression patterns of CD3, CD4, CD8, CD45RO, and FOXP3 at the single cell level. This multiplexed assessment will allow us to examine the epidemiologic and prognostic relevance of numerous metrics of T cell response in CRC. This research will leverage the resources of the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). GECCO-CCFR is a large collaborative effort between observational studies of CRC. We have completed genome-wide germline genotyping and have harmonized epidemiologic data regarding a variety of lifestyle factors and personal characteristics for all participating studies. We are conducting DNA sequencing with a panel of 205 human genes and a small number of bacterial genes in CRC tumor tissue. Prospective follow-up for survival is ongoing, and we have harmonized existing survival data. Through this project, we will add information on T cell response in CRC for >2,500 CRC cases to the GECCO-CCFR resource. This project provides an unprecedented opportunity to investigate the epidemiology of the T cell response in CRC and the relationship of that response with personal and tumor characteristics. Insights gained through this novel study could ultimately inform the development and targeted implementation of emerging immunotherapeutic and immunopreventative strategies.